It has been known for years that some algae and bacteria naturally produce small amounts of hydrogen gas. The limiting factor is the fact that hydrogenase (the enzyme that catalyzes the hydrogen production reaction) is downregulated in the presence of oxygen. Because oxygen is a by-product of photosynthesis, it was necessary to shut down photosynthesis in the alga in an anaerobic environment for the production of greater amounts of hydrogen. This naturally was not a sustainable process, as the algae would initially produce hydrogen in response to the environmental stress and then die in short order. Several attempts were made to try and trick the algae into producing hydrogen without killing them in the process. For example, U.S. Pat. No. 4,442,211 disclosed a process for producing hydrogen by subjecting algae in an aqueous phase to light. Irradiation is increased by culturing algae which has been bleached during a first period of irradiation in a culture medium in a aerobic atmosphere until it has regained color and then subjecting this algae to a second period of irradiation wherein hydrogen is produced at an enhanced rate.
It was later discovered that in the absence of sulfur from the growth media, algae produce hydrogen gas. Sulfur is taken in by the cell through the chloroplasts as sulfate ions. CrcpSulP is a sulfate permease that migrates from the site of transcription in the nucleus to the chloroplasts. Its function as an enzyme is to facilitate sulfate uptake by the chloroplast. Sulfate availability to the chloroplast influences the rate of oxygenic photosynthesis. If the chloroplast is unable to intake an adequate amount of sulfate, then normal oxygen-producing photosynthesis is reduced. If the alga is in a substantially oxygen-free system in the presence of light, it begins photosynthesizing through an alternate cellular pathway, which leads to hydrogen production.
Under oxygenic photosynthesis conditions, and following a dark anaerobic induction, the activity of the hydrogenase is only transient in nature. It lasts from several seconds to a few minutes. This is because photosynthetic O2 is a powerful inhibitor of the [Fe]-hydrogenase (Ghirardi et al. (2000) Trends Biotechnol. 12:506–511) and a positive suppressor of hydrogenase gene expression (Happe and Kaminski (2002) Eur. J. Biochem. 269(3):1022–1032).
Accordingly, there is a need for a process that obviates the need for removing sulfur from the algal growth medium, and thus alleviates cumbersome nutrient removal procedures or adding new algae to the culture. Further, there is a need for a process that permits a continuous and streamlined production of hydrogen from sunlight and water, while alleviating the need for the cells to go back to normal photosynthesis in order to recover lost metabolites such as starch and protein. Further, there is a need to produce hydrogen, making use as broad a portion of the solar spectrum as possible. Thus, there is a need to provide efficient hydrogen production in a closed system using green algae and photosynthetic purple bacteria. Additionally, there is a need for an assay to identify transgenic algae that have decreased ability to uptake sulfate. Using algae to produce hydrogen on a commercial scale has clear advantages for the environment, for reversing the effects of global warming, for decreasing dependence on a limited supply of energy such as oil, and for creating a nearly limitless source of energy. The present invention was developed in an attempt to meet these and other needs.